1. Field of the Invention
This invention relates to an immunoassay method, and to apparatus for carrying out the method. More specifically, the invention relates to an immunoassay method for measurement of the quantity of one or more antigens or antibodies, hereinafter called target antigens or antibodies, in a fluid or tissue specimen. The target antigens or antibodies to be quantitatively determined are reacted with tagged antibodies or antigens which form a complex with the target, and the formed complexes are counted by detection of the tagging elements.
2. Description of the Prior Art
Raddioimmunoassay (RIA) is currently the best developed methodd of immunoassays. A radioactive isotope, usually I-125, is used to label a known antigen (hereinafter called Ag), and the labeled Ag (Ag*) competes with the target Ag of a specimen for the binding site of a given amount of "monomeric" antibody (hereinafter called Ab). The radioactivity of the Ag*-Ab precipitants, often with a second species specific Ab for precipitation, inversely indicates the amount of non-radioactive Ag-Ab complexes in the system, and therefore the amount of the target Ag of the specimen. When instead the antibodies are labeled (Ab*) to directly measure the Ag or ligand, rather than use of Ag* and a limited amount of Ab, the technique is called immumoradiometricassay (IRA).
RIA has been easier to use than IRA due to the difficulties of obtaining monomeric Ab in a consistant purity from an in vivo system that is inherently heterogeneous. Recent progress in monoclonal technology promises to make available the truely monomeric Ab in commercial quantity, and thereby the use of IRA which is a direct and more simple approach. The new method of the invention described herein can be applied either to a direct binding or to a competitive binding assay, and the former direct binding is described for the purpose of illustration.
There are about 5.times.10.sup.16 Ab per ml in a typical animal serum, with each Ab typically having about 20,000 atoms and a molecular weight of about 150,000. If there are 5 million kinds of Ab, each kind would on the average have 10.sup.10 copies per ml. This abundance in copies and richness in variety are what make the immunoassays so easy to prepare and so powerful a tool for measurement.
There are, however, several drawbacks in a method as powerful as RIA. If labeled iodine appears at the sensitive position of the Ag, i.e., the epitope, it would alter the immune affinity. Incubation time for RIA, especially when a second antispecies globulin is used for precipitation, is too long. The presence of agglutinating elements in a specimen such as the serum rheumatoid factor (RF) and the Clq unit of the complement can often give rise to unintended agglutinations. But by far, the most serious problem of the method has been the disposal of voluminous radioactive waste.
In order to avoid the problem of radioactive waste disposal, two recent non-radio immune methods have developed for clinical applications. One method is the homogeneous enzyme immunoassay where an active enzyme is bound to an Ag and the enzyme activity becomes greatly reduced when the enzyme-conjugated Ag submerges to be an integral part of the Ag-Ab complex. This technique precludes the need for separating bound from free, and enables simple colorimetric detection of the enzyme related rate reactions. The sensitivity of the method and the cost of the enzyme bound Ag, however, must be improved in order to be competitive with RIA. Also like RIA, in this emzymatic approach it can be difficult to assay a large number of different Ag-Ab complexes together. Another non-radio method is the fluorescent immunoassay (FIA) where instead of the radio labeled Ag, fluorescent dye-labeled Ag competes with Ag for the binding sites of a limited amount of Ab. The elute of fluorescent Ag-Ab complex can be counted by a fluorometer, which can be automated, just like that of the automated gamma ray counters for RIA. Except for the radiowastes, FIA shares similar drawbacks of RIA, and at a substantially reduced sensitivity. The FIA method is described in Robert E. Curry et al., Clinical Chemistry, Vol. 25, No. 9, (1979),pages 1591-1595.
Since the discoveries of RIA and IRA, a major improvement has been the introduction of solid phase systems. With a solid phase, Ab or Ag are absorbed or covalently conjugated onto a matrix such as cellulose, polystyrene (latex) or polyacrylamide to form an "immobilized system". The solid phase system can be in the form of a wall or beads, can give easy and precise separation of bound from free, requires fewer manipulations, and provides lower non-specific bindings. Other immune assays such as FIA and electrophoretic methods can also use the solid phase systems to take advantage of their inherent ease of bringing reagents into the assay and of separating bound from free.
With a sufficiently small solid phase system, the mobility of the solid phase units can also be exploited. In particle immunoassay (PIA), for example, 0.8 .mu.m latex beads are each coated by 10.sup.5 Ab. The coated beads can react like the Ag-Ab complexes with respect to the agglutinating agents such as RF, Clq, or the murine agglutinators. In a PIA, Ag-Ab complexes compete for the agglutinators with a given amount of the coated beads, while the residual, non-agglutinated beads become proportional to the amount of Ag of the specimen. The beads are counted by their scattering of light. A review of various immune methods can be seen from "Immunoassays for the 80s," edited by Voller et al., University Park Press, Baltimore, Md., 1981. In this publication, PIA is described in an article by P.L. Masson et al. on pages 35-41.
In such known labeled immunoassays, the labeling elements, which can be radioactive or fluorescent, are conjugated to the Ag or Ab and exposed to the assaying chemistry. They cannot easily be separated from the system even with the aid of solid phase units. For PIA, light photons are used to count the single beads discriminately, so that the beads must have a dimension of greater than 0.8 .mu.m, about twice the photon wavelength, in order to be observable. Compared to the size of an Ab, the bead is two to three orders of magnitude larger in its longest linear dimension, so that the thermodynamic and chemical characteristics of the bead are very different from those of the Ab. As a result, agglutinating agents must be used in PIA.